BMSCs from SD rats (4 weeks,60-80g) were
isolated and harvested as described previously[11].In brief, bone marrow tissues were
acquired by flushing the cavities of femurs and tibias using a syringe and
22-gauge needle into culture medium (Dulbecco’s modified Eagle’s medium, Gibco,
Carlsbad, CA, USA ; 10% fetal bovine serum, Hyclone, Logan, UT, USA; 2 mM
L-glutamine; 10,000 U/L penicillin,
and10
mg/L streptomycin, Gibco BRL,
Life Technologies, Paisley, United Kingdom). The whole volume of the flushing
fluid was made into the single-cell suspension and seeded into 15 ml culture
flasks with culture medium. Cells were cultured at 37°C in a humidified
environment with 5% CO2. Non adherent cells were removed 24 hours
later, and adherent cell colonies were washed three times with
phosphate-buffered saline solution. Fresh complete medium was added and changed
every 3-4 days. Cells were subcultured 1:2 or 1:4 when they reached 80-90%
confluence. Cells used in this experiment were all harvested from three
passages. Cell surface markers CD44 were detected by cell immunohistochemistry
for identification of BMSCs.